Chimeric antigen receptor and immune effector cell expressing chimeric antigen receptor

Abstract

Provided are a chimeric antigen receptor capable of targeting B7-H3, a nucleic acid molecule encoding the chimeric antigen receptor, a nucleic acid construct comprising the nucleic acid molecule, an immune effector cell expressing the chimeric antigen receptor, and use thereof. The chimeric antigen receptor comprises an anti-B7-H3 binding domain, a hinge region, a transmembrane domain and a signal transduction domain. Further provided are a composition and a method for diagnosing, treating or preventing tumors that express B7-H3.

1 . A B7-H3-targeting chimeric antigen receptor (CAR), characterized by comprising: an anti-B7-H3 binding domain, a hinge region, a transmembrane domain, and a signal transduction domain;
 preferably, the anti-B7-H3 binding domain comprises an anti-B7-H3 single-chain antibody or antigen-binding portion, and the antibody or antigen-binding portion comprises a light-chain CDR selected from amino acid sequences of SEQ ID NO. 5-7, 17-19, 29-31, 41-43, 53-55, 65-67, 77-79, 89-91, 101-103, 113-115 or any variant thereof; and, comprises a heavy-chain CDR selected from amino acid sequences of SEQ ID NO. 10-12, 22-24, 34-36, 46-48, 58-60, 70-72, 82-84, 94-96, 106-108, 118-120 or any variant thereof.   
     
     
         2 . (canceled) 
     
     
         3 . The CAR according to  claim 1 , wherein the anti-B7-H3 binding domain comprises an anti-B7-H3 antibody or antigen-binding portion, and the antibody or antigen-binding portion comprises a combination of CDRs selected from the following light and heavy chains;
 (1) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 5-7, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 10-12;   (2) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 17-19, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 22-24;   (3) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 29-31, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 34-36;   (4) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 41-43, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 46-48;   (5) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 53-55, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 58-60;   (6) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 65-67, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 70-72;   (7) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 77-79, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 82-84;   (8) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 89-91, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 94-96;   (9) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 101-103, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 106-108; and   (10) light-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 113-115, and heavy-chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 118-120.   
     
     
         4 . The CAR according to  claim 1 , wherein the anti-B7-H3 binding domain comprises an anti-B7-H3 antibody or antigen-binding portion, and the antibody or antigen-binding portion comprises a light-chain sequence selected from amino acid sequences of SEQ ID NO.4, 16, 28, 40, 52, 64, 76, 88, 100, 112 or any variant thereof;
 and, comprises a heavy-chain sequence selected from amino acid sequences of SEQ ID NO. 9, 21, 33, 45, 57, 69, 81, 93, 105, 117 or any variant thereof.   
     
     
         5 . The CAR according to  claim 1 , wherein the anti-B7-H3 binding domain comprises an anti-B7-H3 antibody (scFv) domain; preferably, the anti-B7-H3 single-chain antibody has an amino acid sequence selected from amino acid sequences of SEQ ID NO. 2, 14, 26, 38, 50, 62, 74, 86, 98, 110 or any variant thereof. 
     
     
         6 . The CAR according to  claim 1 , wherein the transmembrane domain is selected from one or more of transmembrane domains of α, β, ζ chains of TCR, CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD3γ, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, PD-1; preferably, the transmembrane domain is selected from one or more of transmembrane domains of CD8α, CD4, CD45, PD-1, CD154, CD28; preferably, the transmembrane domain is selected from one or more of transmembrane domains of CD8α or CD28; more preferably, the transmembrane domain has an amino acid sequence selected from amino acid sequences of SEQ ID NO. 124 or any variant thereof,
 the hinge region is selected from one or more of an extracellular hinge region of CD8, a hinge region of IgG1 Fc CH2CH3, a hinge region of IgD, an extracellular hinge region of CD28, a hinge region of IgG4 Fc CH2CH3, and an extracellular hinge region of CD4; preferably, the hinge region is a hinge region of CD8α; more preferably, the hinge region has an amino acid sequence selected from amino acid sequences of SEQ ID NO. 122 or any variant thereof, 
 preferably, the signal transduction domain is selected from one or more of intracellular signaling regions of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), CD66d, CD3C preferably, the signal transduction domain is selected from an intracellular signaling region of CD33C more preferably, the signal transduction domain has an amino acid sequence selected from amino acid sequences of SEQ ID NO. 130 or any variant thereof; 
 preferably, the signal transduction domain further comprises one or more costimulatory domains; preferably, the costimulatory domain is selected from one or more of intracellular signaling regions of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, 4-1BB, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, 4-1BB; preferably, the costimulatory domain is selected from intracellular signaling regions of 4-1BB, CD134, CD28, OX40; preferably, the costimulatory domain is selected from one or more of intracellular signaling regions of 4-1BB or CD28; more preferably, the costimulatory domain has an amino acid sequence selected from amino acid sequences of SEQ ID NO. 126, 128 or any variant thereof. 
 
     
     
         7 . (canceled) 
     
     
         8 . An isolated nucleic acid molecule, comprising a polynucleotide sequence encoding the CAR according to  claim 1 ; preferably, the nucleic acid molecule has a nucleotide sequence selected from nucleotide sequences of SEQ ID NO. 1, 13, 25, 37, 49, 61, 73, 85, 97, 109 or any variant thereof. 
     
     
         9 . A nucleic acid construct, comprising the nucleic acid molecule according to  claim 8 ; preferably, the nucleic acid construct is a viral vector; more preferably, the viral vector is one or more of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector. 
     
     
         10 . A virus, comprising the nucleic acid molecule according to  claim 8 ; preferably, the virus is one or more of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus. 
     
     
         11 . A method for preparation of a genetically modified immune cell targeting tumor cells expressing B7-H3, comprising: a step of obtaining the CAR according to  claim 1 . 
     
     
         12 . An isolated host cell, expressing the CAR according to  claim 1 ; preferably, the host cell is a mammalian cell; more preferably, the host cell is one or more of a T cell, an NK cell or cell line, a 76T cell, an NKT cell, a macrophage or cell line, a PG13 cell line; more preferably, the host cell is a T cell; the most preferably, the host cell is a primarily cultured T cell. 
     
     
         13 . The host cell according to  claim 12 , wherein the host cell further expresses other effector molecules, the other effector molecules comprise but are not limited to a cytokine, another CAR, a chemokine receptor, an siRNA reducing the expression of PD-1 or a protein blocking PD-L1, a TCR or a safety switch;
 preferably, the cytokine is selected from one or more of TNF-α, TNF-β, VEGF, TPO, NGF-β, PDGF, TGF-α, TGF-β, IGF-I, IGF-II, EPO, M-CSF, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, an interferon, an angiostatin, a thrombospondin, an endostatin;   preferably, the chemokine receptor is selected from one or more of CCR2, CCR5, CXCR2, CXCR4;   preferably, the safety switch is selected from one or more of HSVTK, VZVTK, iCaspase-9, iCaspase-1, iCaspase-8, truncated EGFR, RQR8.   
     
     
         14 . A pharmaceutical composition, comprising the CAR according to  claim 1 . 
     
     
         15 . A method for disease diagnosis, treatment or prevention in a subject, comprising: a step of obtaining the CAR according to  claim 1 , wherein the disease is selected from one or more of cancer, an inflammatory disease, an autoimmune disease;
 preferably, the disease is a tumor expressing B7-H3; more preferably, the disease is selected from one or more of brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, skin cancer, stomach cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, lung cancer, and Kaposi's sarcoma; more preferably, the brain cancer is selected from one or more of glioblastoma, astrocytoma, meningioma, oligodendroglioma, neuroglioma.   
     
     
         16 . A method for preparation of a drug, comprising: a step of obtaining the CAR according to  claim 1 , preferably, the drug is used to diagnose, treat or prevent one or more of cancer, an inflammatory disease, an autoimmune disease;
 preferably, the drug is used to diagnose, treat or prevent a tumor expressing B7-H3; more preferably, the drug is used to diagnose, treat or prevent one or more selected from brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, skin cancer, stomach cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, lung cancer, and Kaposi's sarcoma; more preferably, the brain cancer is selected from one or more of glioblastoma, astrocytoma, meningioma, oligodendroglioma, neuroglioma.   
     
     
         17 . A method for preparation of a genetically modified immune cell targeting tumor cells expressing B7-H3, comprising: a step of obtaining of the nucleic acid molecule according to  claim 8 . 
     
     
         18 . An isolated host cell, comprising the isolated nucleic acid molecule according to  claim 8 ; preferably, the host cell is a mammalian cell; more preferably, the host cell is one or more of a T cell, an NK cell or cell line, a 76T cell, an NKT cell, a macrophage or cell line, a PG13 cell line; more preferably, the host cell is a T cell; the most preferably, the host cell is a primarily cultured T cell. 
     
     
         19 . The host cell according to  claim 18 , wherein the host cell further expresses other effector molecules, the other effector molecules comprise but are not limited to a cytokine, another CAR, a chemokine receptor, an siRNA reducing the expression of PD-1 or a protein blocking PD-L1, a TCR or a safety switch;
 preferably, the cytokine is selected from one or more of TNF-α, TNF-β, VEGF, TPO, NGF-β, PDGF, TGF-α, TGF-β, IGF-I, IGF-II, EPO, M-CSF, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, an interferon, an angiostatin, a thrombospondin, an endostatin;   preferably, the chemokine receptor is selected from one or more of CCR2, CCR5, CXCR2, CXCR4;   preferably, the safety switch is selected from one or more of HSVTK, VZVTK, iCaspase-9, iCaspase-1, iCaspase-8, truncated EGFR, RQR8.   
     
     
         20 . A pharmaceutical composition, comprising the nucleic acid molecule according to  claim 8 . 
     
     
         21 . A method for disease diagnosis, treatment or prevention in a subject, comprising: a step of obtaining a nucleic acid molecule encoding the CAR according to  claim 1 , wherein the disease is selected from one or more of cancer, an inflammatory disease, an autoimmune disease;
 preferably, the disease is a tumor expressing B7-H3; more preferably, the disease is selected from one or more of brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, skin cancer, stomach cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, lung cancer, and Kaposi's sarcoma; more preferably, the brain cancer is selected from one or more of glioblastoma, astrocytoma, meningioma, oligodendroglioma, neuroglioma.   
     
     
         22 . A method for preparation of a drug, comprising: a step of obtaining the nucleic acid molecule according to  claim 8 ; preferably, the drug is used to diagnose, treat or prevent one or more of cancer, an inflammatory disease, an autoimmune disease;
 preferably, the drug is used to diagnose, treat or prevent a tumor expressing B7-H3; more preferably, the drug is used to diagnose, treat or prevent one or more selected from brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, skin cancer, stomach cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, lung cancer, and Kaposi's sarcoma; more preferably, the brain cancer is selected from one or more of glioblastoma, astrocytoma, meningioma, oligodendroglioma, neuroglioma.